Gliomas are genotypically heterogeneous, both in biomarker expression (or lack thereof) and in their ability to support replication of genetically modified herpes simplex virus (HSV) vectors. In addition to heterogeneity in Gene Expression Profiles (GEP), a subpopulation of glioma cells has been identified that has properties resembling those of neural stem cells and these cells are believed to be responsible for glioma recurrence and to exclusively maintain the neoplastic clone. This subpopulation of cells, referred to as Brain Tumor-lnitiating Cells or Glioma Progenitor Cells (GPC), contribute significantly to chemoresistance and radioresistance of malignant gliomas. These GPC may also display differential susceptibility to oncolysis by genetically engineered HSV alone or combined with radiation. Anti-tumor activity of genetically engineered HSV vectors is dependent on the ability of the host tumor cell to support HSV infection and replication. A clear understanding of the mechanisms of enhanced tumor killing mediated by both y{1}34.5-deleted HSV and receptor-targeted wild-type HSV vectors is critical to achieve improved clinical efficacy. We have already shown that IL-12 expression in a Ay[1}34.5 HSV background results in enhanced anti-tumor activity and this enhanced effect is immune-mediated. The question remains whether this benefit would also be seen with a targeted virus in which the virus'ability to overcome the PKR response remains intact or is restored. Project 3 has three fundamental aims. Aim 1 will ascertain the relative oncolytic potential of each of the novel mutant HSV engineered by Projects 1 and 2 to use different entry mechanisms or to overcome innate host responses. Their antitumor activity on glioma cells, alone or combined with irradiation, will be compared with that of our current Ay{1}34.5 HSV vectors. These studies will (i) determine which modified viruses are best able to infect cells and overcome natural host defenses, and, (ii) lead to the engineering of viruses that combine these optimal features. Aim 2 will focus on defining the comparative susceptibility of a subpopulation of glioma progenitor cells to oncolytic Ay{1}34.5 HSV alone and with irradiation. HSV specifically targeted to CD133 will be tested for efficacy on GPC while retaining safety for normal neuroglial cells. GEP of GPC and non-GPC tumor cells will be compared to reveal potential causes for resistance to HSV. Aim 3 will complete preclinical testing of genetically engineered HSV that incorporate all genetic modifications.